Cell flow apparatus and method for real-time of cellular responses
First Claim
1. A method for identifying compounds which produce a cellular response comprising:
- (a) using an automated and computer controlled apparatus to sequentially combine each of a plurality of cell suspensions with one or more test compounds to sequentially form each of a plurality of test mixtures;
(b) sequentially directing each of said plurality of test mixtures through a detection zone, the detection zone being capable of detecting a plurality of cellular responses simultaneously; and
(c) simultaneously measuring a plurality of cellular responses to the one or more test compounds in said suspended cells as each of said plurality of test mixtures are flowing through said detection zone.
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Accused Products
Abstract
An apparatus and method for real-time measurement of a cellular response of a test compound or series of test compounds (303) on a flowing suspension of cells (349), in which a homogeneous suspension of each member of a series of cell types (349) is combined with a concentration of a test compound (303), directed through a detection zone (355), and a cellular response of the living cells is measured in real time as the cells in the test mixture are flowing through the detection zone (355). The apparatus may be used in automated screening of libraries of compounds, and is capable of real-time variation of concentrations of test and standard compounds and generation of dose/response profiles within a short time span.
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Citations
84 Claims
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1. A method for identifying compounds which produce a cellular response comprising:
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(a) using an automated and computer controlled apparatus to sequentially combine each of a plurality of cell suspensions with one or more test compounds to sequentially form each of a plurality of test mixtures;
(b) sequentially directing each of said plurality of test mixtures through a detection zone, the detection zone being capable of detecting a plurality of cellular responses simultaneously; and
(c) simultaneously measuring a plurality of cellular responses to the one or more test compounds in said suspended cells as each of said plurality of test mixtures are flowing through said detection zone. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 34, 35, 36, 37, 38, 39, 72, 73, 74, 78)
(d) combining a suspension of said cells with one or more standard compounds having a known effect on said cellular response of said cells to form standard mixtures;
(e) directing the standard mixtures through the detection zone; and
(f) measuring the cellular response of said cells to said one or more standard compounds.
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35. The method of claim 1, wherein one or more standard compounds and said one or more test compounds are simultaneously mixed with each of said cell suspensions, and said measuring step detects whether said one or more test compounds alter a known effect of said one or more standard compounds.
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36. The method of claim 35, wherein said one or more standard compounds is an antagonist of said cellular response.
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37. The method of claim 35, wherein said one or more standard compounds is an agonist of said cellular response.
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38. The method of claim 35, wherein said cell suspensions comprise more than one cell type.
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39. The method of claim 35, wherein the step of directing said test mixtures through a detection zone comprises directing said test mixtures through a flow cytometer.
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72. The method of claim 1, wherein a range of concentrations of said test compounds is combined with said cell suspensions.
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73. The method of claim 1, further comprising combining one or more compounds which produce a known cellular response with said plurality of cell suspensions and said test compounds.
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74. The method of claim 73, wherein a range of concentrations of said one or more compounds which produce a known cellular response is combined with said plurality of cell suspensions and said test compounds.
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78. The method of claim 1, wherein a range of concentrations of said agents capable of binding said molecules is combined with said samples.
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9. A method for identifying compounds which produce a cellular response comprising:
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(a) using an automated and computer controlled apparatus to sequentially combine each of a plurality of cell suspensions with one or more test compounds to form each of a plurality of test mixtures;
(b) sequentially directing each of said plurality of test mixtures through a detection zone, said detection zone being capable of detecting cellular responses in individual cells; and
(c) measuring one or more cellular responses to said one or more test compounds in individual suspended cells as each of said plurality of test mixtures are flowing through said detection zone. - View Dependent Claims (10, 11, 12, 13, 14, 15, 75, 76, 77)
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16. A method of determining whether a sample contains one or more molecules comprising:
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using an automated and computer controlled apparatus to sequentially combine one or more agents capable of binding one or more molecules with each of a plurality of samples to form a plurality of test mixtures;
sequentially directing each of said plurality of test mixtures through a detection zone; and
determining whether said one or more agents are bound to said one or more molecules as each of said test mixtures are flowing through said detection zone. - View Dependent Claims (17, 18, 19, 20, 21)
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22. A method of determining whether a sample contains one or more polypeptides comprising:
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attaching one or more antibodies which specifically bind said one or more polypeptides to solid supports;
using an automated and computer controlled apparatus to sequentially combine said one or more antibodies attached to solid supports with samples to form a plurality of test mixtures;
sequentially directing each of said plurality of test mixtures through a detection zone; and
determining whether said one or more antibodies are bound to said one or more polypeptides as each of test mixtures are flowing through said detection zone. - View Dependent Claims (23, 24, 25)
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26. A method of determining whether a sample contains one or more nulceic acids comprising:
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attaching one or more nucleic acid probes which specifically bind said nucleic acids to solid supports;
using an automated and computer controlled apparatus to sequentially combine said one or more probes attached to said solid supports with samples to form a plurality test mixtures;
sequentially directing each of said plurality of test mixtures through a detection zone; and
determining whether said one or more probes are bound to said one or more nucleic acids as each of said test mixtures are flowing through said detection zone. - View Dependent Claims (27, 28, 29)
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30. A method of determining whether a sample contains one or more single nucleotide polymorphisms comprising:
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performing an extension reaction on a each of a plurality of nucleic acid samples using one or more nucleic acid primers for each of said plurality of nucleic acid samples, said one or more nucleic acid primers having a 3′
end immediately adjacent to the polymorphic base of one or more single nucleotide polymorphisms, thereby incorporating one base into said nucleic acid primers;
attaching the extended primers to solid supports;
sequentially directing said solid supports having primers attached thereto for each of said plurality of nucleic acid samples through a detection zone in an apparatus; and
determining the identities of the bases incorporated in said extension reaction. - View Dependent Claims (31, 32, 33)
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40. A method of obtaining cells having a desired phenotype or cellular response profile comprising the steps of:
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(a) using an automated and computer controlled apparatus to sequentially combine a each of a plurality of cell suspensions with one or more compounds which produce a cellular response to form a plurality of test mixtures;
(b) sequentially directing each of said plurality of test mixtures through a detection zone, said detection zone being capable of detecting a plurality of cellular parameters simultaneously;
(c) simultaneously measuring a plurality of cellular parameters in said suspended cells as each of said plurality of test mixtures are flowing through said detection zone; and
(d) delivering cells having a desired phenotype or cellular response profile into a receptacle. - View Dependent Claims (79, 80, 81)
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41. A method of obtaining cells having a desired phenotype or cellular response profile comprising the steps of:
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(a) using an automated and computer controlled apparatus to sequentially combine a each of a plurality of cell suspensions with one or more compounds which produce a cellular response to form a plurality of test mixtures;
(b) sequentially directing each of said plurality of test mixtures through a detection zone, said detection zone being capable of detecting cellular responses in individual cells;
(c) simultaneously measuring a plurality of cellular parameters in said suspended cells as each of said plurality of test mixtures are flowing through said detection zone; and
(d) delivering cells having a desired phenotype or cellular response profile into a receptacle. - View Dependent Claims (82, 83, 84)
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42. An apparatus comprising:
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an automated test compound source capable of sequentially providing each of a plurality of test compounds;
an automated test substrate source capable of sequentially providing each of a plurality of test compounds;
a mixing chamber in fluid communication with said test compound source and said test substrate source, wherein said mixing chamber is adapted to combine a test compound received from said test compound source with a test substrate received from said test substrate source to generate a mixture; and
a detector in fluid communication with said mixing chamber, said detector being capable of detecting an interaction between said test compound and said test substrate while said mixture is passing through said detector. - View Dependent Claims (43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 66, 67, 68)
said test compound source comprises one or more sample inputs for sequentially providing each of a plurality of samples, each of said samples comprising one or more test compounds to be evaluated for the ability to produce a cellular response or a solution to be evaluated for the presence of molecules;
said test substrate source comprises one or more cell or particle inputs for sequentially providing a each of a plurality of cell suspensions or particles;
said mixing chamber receives the samples from said test compound source, receives the cell suspensions or particles from the cell or particle input and mixes each sample with the cell suspension or particles; and
said detector is capable of detecting a plurality of signals simultaneously such that said detector can simultaneously measure a plurality of cellular responses in the suspended cells or simultaneously determine whether a plurality of molecules are present in said samples.
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44. The apparatus of claim 43 wherein said detector detects a signal from a single cell in said cell suspension or from a single particle.
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45. The apparatus of claim 43 wherein said detector is a flow cytometer.
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46. The apparatus of claim 43, further comprising a coupler disposed between said mixing zone and said detector.
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47. The apparatus of claim 46, wherein said coupler delivers slugs comprising said samples and said cell suspension or particles to said detector.
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48. The apparatus of claim 47, wherein said coupler delivers substantially undiluted slugs to said detector.
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49. The apparatus of claim 43, wherein said detector delivers cells having a desired phenotype or cellular response to a receptacle.
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50. The apparatus of claim 43 wherein said one or more sample inputs comprise an automated robotic sampler capable of sequentially selecting each of a plurality of specified test compounds from a library of test compounds.
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51. The apparatus of claim 50 further comprising:
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a controller, coupled to said one or more sample inputs, for controlling the operation of the automated robotic sampler; and
a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, thereby controlling the selection and retrieval of test compounds by said one or more sample inputs from said test compound library.
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52. The apparatus of claim 51 further comprising a gradient pump having an input and an output, coupled to said sample input, for adjusting the concentration level of said test compound transferred to said mixing zone from said sample input, wherein:
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said sample input comprises;
a first intake nozzle for receiving each of the said specified test compounds;
a second intake nozzle for receiving a buffer solution; and
wherein said gradient pump is coupled to the first and second intake nozzles and receives specified concentrations of each of the specified test compounds by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound.
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53. The apparatus of claim 52 further comprising a second pump, coupled to an input of said mixing zone, for pumping said suspension of cells or said particles from said cell or particle input into the mixing zone.
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54. The apparatus of claim 53 further comprising a reaction developing line, having an input coupled to an output of said mixing zone and an output in fluid communication with said detector wherein said reaction developing line provides a reaction time delay for a mixture received from the mixing zone.
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55. The apparatus of claim 54 further comprising:
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a pump, coupled to the output of said detector, for providing negative pressure to the apparatus;
a proportionating valve, coupled to said sample input, for adjusting the concentration level of said test compound transferred to said mixing zone from said one or more sample inputs, wherein said one or more sample inputs further comprise;
a first intake nozzle for receiving each of said specified test compounds;
a second intake nozzle for receiving a buffer solution; and
the proportionating valve receives specified concentrations of each of the specified test compounds by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound.
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56. The apparatus of claim 43 further comprising a plurality of cell suspension reservoirs.
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57. The apparatus of claim 43 further comprising a standard compound sampler, coupled to said mixing zone, for providing one or more standard compounds having a known effect on said cellular response of said suspended cells or a known interaction with said particles, wherein said mixing zone receives said one or more standard compounds from the standard compound sampler and mixes said one or more standard compounds with the suspended cells or particles and said detector measures the cellular response of the suspended cells to said one or more standard compounds or the interaction between said one or more standard compounds and said particles.
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58. The apparatus of claim 57 wherein said mixing zone simultaneously mixes said sample and said one or more standard compounds with said suspended cells or said particles and said detector detects said known effect or an alteration of said known effect on the cellular responses of said suspended cells or said known interaction between said standard compound and said particles or an alteration of said known interaction between said standard compound and said particles.
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59. The apparatus of claim 58 wherein said standard compound sampler is an automated robotic sampler capable of selecting a specified standard compound from a library of standard compounds.
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66. The apparatus of claim 42, wherein said test compound source is capable of providing said test compounds at a single concentration or over a range of concentrations.
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67. The apparatus of claim 42, further comprising a known compound source capable of sequentially providing each of a plurality of compounds which produce a known cellular response, said known compound source being in fluid communication with said mixing chamber such that said mixing chamber can combine a test compound, a compound which produces a known cellular response, and a cell suspension.
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68. The apparatus of claim 67, wherein said known compound source is capable of providing the compounds which produce a known cellular response at a single concentration or over a range of concentrations.
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60. An apparatus comprising:
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one or more automated sample inputs for sequentially providing each of a plurality of samples, said each of said samples comprising one or more test compounds to be evaluated for the ability to produce a cellular response or a solution to be evaluated for the presence of molecules;
one or more automated cell or particle inputs for sequentially providing each of a plurality of cell suspensions or particles;
a mixing zone, coupled to said sample input, for receiving the samples, receiving the cell suspension or particles from the cell or particle input and mixing each sample with the cell suspensions or particles; and
a detector which detects one or more signals from a single cell or particle, said detector being coupled to the mixing zone and being capable of measuring one or more cellular responses in the suspended cells or determining whether one or more molecules are present in said samples. - View Dependent Claims (61, 62, 63, 64, 65, 69, 70, 71)
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Specification